Figure 1. Identification of the in vivo phosphorylation sites on hCdc34 and yCdc34.

(A) HEK-293 cells were transiently transfected with either pCMV-Tag2 (vector) or pCMV-Tag2-hCdc34(Wt) and labelled with [³²P]P_i. Human Cdc34 was immunoprecipitated, separated by SDS/PAGE and visualized by autoradiography. (B) Wild-type ³²P-labelled hCdc34 (left panel) or its mutant derivative S231T (right panel) were subjected to phospho amino acid analysis. The positions of phosphoserine (Ser∼P) and phosphothreonine (Thr∼P) are indicated. (C) HEK-293 cells were transiently transfected with either pCMV-Tag2 (vector) or pCMV-Tag2 constructs expressing wild-type hCdc34 or its mutant derivatives S(all4)A (S203A/S222A/S231A/S236A), S203 (S222A/S231A/S236A), S222 (S203A/S231A/S236A) and S236 (S203A/S222A/S231A) and subjected to the same treatment as in (A). After SDS/PAGE, proteins were visualized by autoradiography (³²P) or by immunoblotting (IB) with an anti-hCdc34 antibody. (D) Yeast cells (W303-1A) were transformed with empty pESC-TRP (vector) or pESC1 constructs expressing either yeast wild-type Cdc34 (yCdc34) or its C-terminal truncation derivatives 252Δ (residues 1–252) and 200Δ (residues 1–200). After labelling with [³²P]P_i, cells were lysed, and yCdc34 was immunoprecipitated, separated by SDS/PAGE and visualized by autoradiography (³²P) or by immunoblotting (IB) with anti-FLAG antibody. The asterisk indicates the position of the light immunoglobulin chain of the anti-FLAG beads. (E) Yeast cells (W303-1A) were transformed with empty pESC-TRP (vector) or pESC1 constructs expressing either yeast wild-type Cdc34 (yCdc34) or its mutant derivatives S(all6)A (S207A/S216A/S263A/S268A/S282A/S292A), S207 (S216A/S263A/S268A/S282A/S292A), S216 (S207A/S263A/S268A/S282A/S292A), S263 (S207A/S216A/S268A/S282A/S292A), S268 (S207A/S216A/S263A/S282A/S292A), S282 (S207A/S216A/S263A/S268A/S292A) and S292 (S207A/S216A/S263A/S268A/S282A). Cells were subjected to metabolic ³²P labelling as in (D), and proteins were visualized by autoradiography (³²P) or by immunoblotting (IB) with an anti-Cdc34 antibody.